Zinc gambogic acid compound as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of a gambogic acid zinc compound, which comprises the following steps: mixing zinc ions and gambogic acid in a first organic solvent under a stirring condition to obtain a first mixture; adding triethylamine and polyvinylpyrrolidone into the first mixture, and stirring for a period of time to obtain a second mixture; and centrifugally separating the precipitate in the second mixture, washing the obtained solid for multiple times, and carrying out vacuum drying to obtain the zinc gambogic acid compound (GAZn-coated PEG NPs). According to the GAZn coated PEG NPs disclosed by the invention, the water solubility and bioavailability of the GA are remarkably improved, the immune activation capability of the GA is further enhanced, and antigen presentation and maturation of dendritic cells can be promoted; the combined action of GA and Zn < 2 + > successfully activates a cGAS-STING pathway, and further promotes the maturation of dendritic cells, so that the expression of HSP-90 can be obviously reduced when the polypeptide is combined with an HIFU technology, the heat tolerance of tumors is inhibited, the treatment effect is enhanced, and the obvious treatment effect is shown.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a zinc gamboge compound and a preparation method and application thereof. Background Art

[0002] In recent years, high-intensity focused ultrasound (HIFU) technology has received widespread attention in the field of gynecological disease treatment. HIFU treats solid tumors by focusing high-intensity ultrasound waves on the target, effectively reducing the incidence of serious adverse reactions. On this basis, phased high-intensity focused ultrasound (pHIFU) technology came into being. Compared with traditional HIFU, it has higher treatment accuracy and energy control accuracy through technical advantages such as phased acoustic beams, electronically controlled multi-focus, and flexible acoustic beam paths. pHIFU technology has shown advantages such as short treatment time and fewer complications in the treatment of benign uterine tumors, providing new possibilities for the future treatment of malignant gynecological tumors.

[0003] HIFU technology can not only induce apoptosis and necrosis of tumor cells, but also produce tumor-specific antigens and activate the immune system. However, the expression of heat shock protein 90 (HSP-90) in the tumor site increases after hyperthermia, which may weaken the therapeutic effect. In addition, the risk of cancer recurrence and metastasis after HIFU technology treatment still exists, because local tumor ablation induced by HIFU technology alone is not sufficient to eliminate all residual cancer cells.

[0004] Garcinia acid (GA) is a compound with antitumor activity extracted from Garcinia resin. As a natural HSP-90 inhibitor, GA shows significant inhibitory effects on a variety of malignant tumor cells. GA induces apoptosis of cancer cells by activating T lymphocytes, showing great potential in cancer prevention and tumor immunotherapy. However, the oral and intravenous bioavailability of GA is low, and it may cause a series of adverse reactions, which limits its clinical application.

[0005] Zinc ions have received attention for their critical role in the integrity of the immune system. Zinc plays a key supporting role in the maintenance, development and activation of immune cells and can also regulate the function of T cells. Zinc deficiency impairs the activity of natural killer cells (NK cells) and cytotoxic T lymphocytes, which play a vital role in fighting viruses, bacteria and tumor cells.

[0006] In summary, the development of a gambogic acid zinc complex aims to improve the bioavailability of GA, reduce its toxicity, and utilize the immunomodulatory effect of zinc ions to enhance the effect of HIFU technology, which is of great significance for improving the therapeutic effect of malignant tumors. The present invention provides a gambogic acid zinc complex and its preparation method and application, which provides a new strategy for the comprehensive treatment of malignant tumors. Summary of the invention

[0007] The purpose of the present invention is to provide a zinc gamboge compound and a preparation method and application thereof in view of the deficiencies in the prior art.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention is to provide a method for preparing a zinc garcinate complex, the steps comprising:

[0010] S1. Mixing zinc ions and gambogic acid in a first organic solvent under stirring to obtain a first mixture;

[0011] S2, adding triethylamine and polyvinyl pyrrolidone to the first mixture, stirring for a period of time to obtain a second mixture;

[0012] S3, separating the precipitate in the second mixture by centrifugation, washing the obtained solid for multiple times and then vacuum drying to obtain the gambogic acid zinc complex.

[0013] Preferably, in the step S1, under the stirring condition, the first solution containing the zinc ions is added dropwise to the second solution containing the gambogic acid to obtain the first mixture;

[0014] Wherein, the solvents of the first solution and the second solution are both the first organic solvent.

[0015] Preferably, in step S1, the stirring conditions include: a stirring speed of 1000-2000 rpm.

[0016] Preferably, in step S1, the molar ratio of the zinc ion to the gambogic acid is 1:(2-6).

[0017] Preferably, in step S1, the first organic solvent is ethanol.

[0018] Preferably, in step S2, the stirring time is 1-3 hours.

[0019] Preferably, in step S3, the centrifugal conditions include: the centrifugal force is 3500g-4500g, and the centrifugal time is 2min-7min.

[0020] Preferably, the steps further include:

[0021] S4, modifying the surface of the solid obtained in step S3 with polyethylene glycol.

[0022] Preferably, the step S4 comprises:

[0023] S4-1, dispersing the solid obtained in step S3 in a second organic solvent, adding a third solution containing 1,2-dioleoyl-sn-glycero-3-phosphate, and performing ultrasonic treatment;

[0024] S4-2, separating the gamboge acid zinc complex coated with 1,2-dioleoyl-sn-glycero-3-phosphate obtained in step S4-1 by centrifugation, washing it multiple times, dispersing it in the fourth solution, and stirring it at a certain stirring speed for a period of time to obtain a third mixture;

[0025] Wherein, the solute of the fourth solution includes: cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-5000;

[0026] S4-3, removing the solvent in the third mixture by rotary evaporation to obtain a zinc gamboge complex with surface modified with polyethylene glycol.

[0027] Preferably, in step S4-1, the second organic solvent is ethanol.

[0028] Preferably, in step S4-1, the solvent of the third solution is a third organic solvent.

[0029] Preferably, in step S4-1, the conditions of the ultrasonic treatment include: the ultrasonic power is 50w-150w, and the ultrasonic time is 10min-20min.

[0030] Preferably, in step S4-2, the centrifugal conditions include: a centrifugal force of 500g-1500g, and a centrifugal time of 2min-7min.

[0031] Preferably, in step S4-2, the solvent of the fourth solution is a fourth organic solvent.

[0032] Preferably, in step S4-2, the stirring speed is 1000-2000 rpm, and the stirring time is overnight.

[0033] Preferably, in step S4-3, the conditions for rotary evaporation include: providing a vacuum negative pressure environment, the temperature of rotary evaporation is 35°C-40°C, the speed of rotary evaporation is 50-100 rpm, and the time of rotary evaporation is 10min-20min.

[0034] Preferably, in step S4-1, the third organic solvent is chloroform.

[0035] Preferably, in step S4-2, the fourth organic solvent is chloroform.

[0036] The second aspect of the present invention is to provide a zinc garcinate complex prepared by the above-mentioned preparation method.

[0037] The third aspect of the present invention is to provide a use of the aforementioned gambogic acid zinc complex in the preparation of a drug for treating cervical cancer.

[0038] The fourth aspect of the present invention is to provide a use of the aforementioned zinc gambogic acid complex in the preparation of a system for treating cervical cancer, wherein the system can administer a therapeutically effective amount of the zinc gambogic acid complex to a patient in combination with high-intensity focused ultrasound.

[0039] Preferably, the system comprises: a first module for administering a therapeutically effective amount of the zinc gamboge acid complex to a patient, a second module for applying high intensity focused ultrasound to the patient, and a control module for controlling the first module and the second module;

[0040] Wherein, the first module, the second module, and the control module are three devices respectively; or,

[0041] The first module, the second module, and the control module are three components of a device respectively.

[0042] Preferably, the high-intensity focused ultrasound is phased high-intensity focused ultrasound.

[0043] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0044] The gambogic acid zinc complex (GAZn-PEG NPs) of the present invention is prepared by combining gambogic acid (GA) with zinc ions (Zn 2+ ) complexed and coated with a lipid bilayer modified with polyethylene glycol (PEG). GAZn-PEG NPs significantly improved the water solubility and bioavailability of GA, and the immune activation ability of GA was further enhanced, which could promote antigen presentation and maturation of dendritic cells; GA and Zn 2+ The combined action of the two successfully activated the cyclic guanosine monophosphate-adenylate synthetase (cGAS)-stimulated interferon gene (STING) pathway, further promoting the maturation of dendritic cells, which can significantly reduce the expression of HSP-90 when combined with HIFU technology, thereby inhibiting the thermal tolerance of the tumor, enhancing the therapeutic effect, and showing a significant therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The middle figure (a) is a schematic diagram of the synthesis process and structure of GAZn-PEG nanoparticles, and the figure (b) is a schematic diagram of the biological response induced by the combined treatment of GAZn-PEG nanoparticles and HIFU;

[0046] Figure 2 Detailed characterization and molecular dynamics analysis of GAZn-PEG nanoparticles; Figure (a) is a transmission electron microscope (TEM) image; Figure (b) is a scanning transmission electron microscope (STEM) element mapping image; Figure (c) is an energy dispersive X-ray spectroscopy (EDS) spectrum; Figure (d) is an X-ray diffraction (XRD) spectrum; Figure (e) is the hydrodynamic diameter of GAZn nanoparticles and GAZn-PEG nanoparticles; Figure (f) is the seven-day stability of GAZn-PEG NPs in different media; Figures (g) to (h) are ultraviolet-visible-near-infrared (UV-vis-NIR) spectral analysis; Figures (i) to (m) are molecular dynamics simulation results, including: GA-Zn 2+ root mean square deviation (RMSD), solvent accessible surface area (SASA), intermolecular hydrogen bonding analysis, detailed schematic diagram of hydrogen bonding and electrostatic interactions inside GAZn NPs, and binding energy analysis of the composites;

[0047] Figure 3 Figure 2 is the in vivo immune response induced by GAZn-PEG NPs; Figure (a) and Figure (b) are flow cytometry and quantitative analysis of mature dendritic cells (DCs) in mouse tumors after different treatments; Figure (c) is the confocal microscopy detection of CD11c in tumors after GAZn-PEG NPs treatment. + Cell infiltration (scale bar 20 μm); Figures (d) to (f) are enzyme-linked immunosorbent assay (ELISA) to evaluate the concentrations of IL-6, IL-12p70, and TNF-α in tumors; Figures (g) to (q) are flow cytometry analysis or statistical analysis of different cells, respectively;

[0048] Figure 4 The inhibitory effect of pHIFU treatment based on GAZn-PEG NPs on tumor growth and the extension of survival of U14 tumor-bearing mice; Figure (a) is a schematic diagram of the tumor challenge and treatment experiment design; Figures (b) to (e) are the response images of tumors in different groups of mice, the average tumor growth, individual tumor growth curves, and survival rates, respectively; Figure (f) shows the H&E staining and HSP-90 immunohistochemistry results of the tumors in the treated and untreated groups on the second day.

[0049] Figure 5Figure 2 is the anti-tumor immune effect of GAZn-PEG NPs+HIFU combined therapy; Figure (a) and Figure (b) are the flow cytometry images and quantitative analysis of mature dendritic cells (DCs), respectively; Figures (c) to (g) and Figures (i) to (l) are the flow cytometry analysis images and quantitative analysis of different cells, respectively; Figure (h) is the confocal microscopy detection of CD4 in tumors after GAZn-PEG NPs+HIFU combined therapy. + and CD8 + T cell infiltration (blue represents DAPI-stained cell nuclei; green represents FITC-labeled anti-CD8 monoclonal antibody (mAb)-stained CD8 + cells; red indicates CD4 stained with PE-labeled anti-CD4 mAb + Cells. Scale bar is 50 μm); Figures (o) to (p) are the secretion levels of TNF-α and IFN-γ in U14 tumors of mice in different treatment groups analyzed by ELISA kit;

[0050] The data are mean ± standard error (SEM). Figure 3 and Figure 5 In, n = 6, statistical significance was analyzed by one-way analysis of variance (ANOVA); Figure 4 In the figure, n=8, the statistical significance of Figure (c) was determined by two-tailed Student's t test, and the statistical significance of Figure (e) was determined by log-rank (Mantel-Cox) test. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns indicates not significant. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention will be described in detail below.

[0052] Unless otherwise defined, technical or scientific terms used in the claims and the specification shall have the common meanings understood by persons having ordinary skills in the technical field to which the present invention belongs.

[0053] The words "include" or similar words used in the patent application specification and claims of the present invention mean that the items appearing before "include" include the items listed after "include" or their equivalents, and do not exclude other items.

[0054] The numerical values ​​mentioned in the present invention include all numerical values ​​that increase from low to high by one unit, assuming that there are at least two units between any lower value and the higher value. For example, if a component or a physical quantity is said to be from 1 to 100, 10 to 90 is more preferred, and 20 to 80 is the most preferred, it is intended to express that values ​​such as 5 to 95, 14 to 76, 23 to 67, 32 to 58, 41 to 49 are clearly listed in this specification; for values ​​less than 1, 0.0001, 0.001, 0.01 or 0.1 are considered to be more suitable as a unit. The above examples are for illustrative purposes only. In fact, all numerical combinations between the lowest value and the highest value listed are deemed to be clearly listed in this specification in a similar manner.

[0055] Example 1

[0056] like Figure 1 As shown, this embodiment provides a gambogic acid zinc complex and a preparation method thereof, and the preparation steps include:

[0057] S1. At a stirring speed of 1500 rpm, 3 mL of an ethanol solution containing 0.0625 mmol of zinc chloride (ZnCl2) was added dropwise to 3 mL of an ethanol solution containing 0.5 mmol of gambogic acid (GA) to obtain a first mixture;

[0058] S2, adding 2 μL of triethylamine and 2 mg of polyvinylpyrrolidone (PVP) to the first mixture, and stirring at room temperature for 2 hours to obtain a second mixture;

[0059] S3, separating the precipitate in the second mixture by centrifugation (4000 g, 5 min), washing the obtained solid with ethanol three times and then vacuum drying at 75° C. to obtain gambogic acid zinc complex (GAZn);

[0060] S4, modifying the surface of the GAZn with polyethylene glycol:

[0061] S4-1, after dispersing 10 mg of the GAZn in 15 mL of ethanol, add 1 mL of a chloroform solution containing 1 mg of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and perform ultrasonic treatment at a power of 100 W for 15 min;

[0062] S4-2, separating the DOPA-coated GAZn obtained in step S4-1 by centrifugation (1000 g, 5 min), washing it three times with 15 mL of anhydrous ethanol, dispersing it in the fourth solution, and stirring it overnight at a stirring speed of 1500 rpm to obtain a third mixture;

[0063] The fourth solution is 15 mL of a chloroform solution containing 9.3 mg of cholesterol, 27 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and 15 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-5000 (DSPE-PEG5000);

[0064] S4-3. The solvent chloroform in the third mixture is removed by rotary evaporation at 80 rpm for 15 min in a water bath at 37° C. under vacuum negative pressure to obtain gamboge zinc complexes (GAZn-PEG NPs) modified with polyethylene glycol on the surface.

[0065] By transmission electron microscopy (TEM) imaging, we observed that GAZn and GAZn-PEG NPs exhibited a uniform spherical morphology with a diameter of approximately 110 nm (see Figure 2 a). Further using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) technology, we obtained the elemental distribution map of GAZn-PEG NPs, which clearly revealed the presence of nitrogen (N), oxygen (O), phosphorus (P) and zinc (Zn) elements (see Figure 2 b). The distribution of these elements was subsequently verified by energy dispersive X-ray spectroscopy (EDS) (see Figure 2 c) X-ray diffraction (XRD) analysis results showed that GAZn-PEG NPs have an amorphous structure, which is consistent with the TEM observation results (see Figure 2 d).

[0066] Dynamic light scattering (DLS) technique was used to measure the hydrated diameters of GAZn NPs and GAZn-PEG NPs, which were 105 nm and 120 nm, respectively (see Figure 2 e). Due to the successful modification with polyethylene glycol (PEG), GAZn-PEG NPs exhibited remarkable stability in cell culture medium containing phosphate buffered saline (PBS), RPMI 1640, or 10% serum, and remained stable for up to seven days of incubation (see Figure 2 f).

[0067] We also measured the UV-NIR absorption spectra of GAZn-PEG NPs at different concentrations and found that there was an approximately linear relationship between the absorbance and the concentration of GAZn-PEG NPs at 360 nm (see Figure 2 h), which showed its good dose-response characteristics.

[0068] In order to gain a deeper understanding of the self-assembly mechanism of GA and zinc chloride (Zn2+) in ethanol solution, molecular dynamics (MD) simulations were performed. The root mean square deviation (RMSD) results showed that all molecules tended to stabilize after 20 nanoseconds (see Figure 2 i). The solvent accessible surface area (SASA) value is a key indicator for evaluating the stability and compactness of a molecular system. The SASA results show that GA and Zn2+ form stable molecular aggregates (see Figure 2 j).

[0069] The intermolecular interactions of the molecular aggregates were further analyzed. In the simulation system, the number of hydrogen bonds between GA-Zn2+ molecules increased rapidly and stabilized after 20 nanoseconds, indicating that the two molecules had aggregated to form a relatively stable nanocluster structure (see Figure 2 k). In-depth analysis of the interactions between the GA-Zn2+ molecules at the center of the nanocluster revealed that the oxygen (O) atoms on the GA-Zn2+ molecules formed hydrogen bonds with the surrounding molecules, promoting their self-assembly (see Figure 2 l). Binding energy analysis shows that electrostatic energy is the main driving force for self-assembly during the entire GA-Zn2+ molecular self-assembly process (see Figure 2 m).

[0070] The MD simulation results clearly show that after 50 ns of molecular dynamics simulation, GA-Zn2+ molecules mainly promote self-assembly through intermolecular hydrogen bonds to form a stable spherical nanocluster structure.

[0071] Example 2

[0072] In vivo experiments showed that GAZn-PEG nanoparticles effectively enhanced the immune response and improved the efficacy of pHIFU in the treatment of cervical cancer by activating the cGAS-STING pathway, promoting dendritic cell maturation, and inhibiting HSP-90 expression.

[0073] In the BALB / c mouse U14 tumor model, we further investigated the effects of GAZn-PEG nanoparticles in immunotherapy and targeted cancer therapy. The mice were randomly divided into five groups and received PBS, zinc chloride (Zn 2+ , 1.2mg / kg), garcinia acid (GA, 2mg / kg), GA and Zn 2+ On the sixth day, the mice were euthanized and tumor tissues were extracted for analysis.

[0074] Flow cytometry results showed that compared with the control group treated with PBS alone, Zn 2+Mice treated with PBS or GA showed a slight increase in the maturation of dendritic cells (DCs). The DCs in the GAZn-PEG-treated group were significantly more mature than those in the other treatment groups, and were significantly higher than those in the PBS, GA, and Zn groups, respectively. 2+ The results of the 2014 study showed that the 10-fold increase in the 40-mg / kg group was 37.6%, 27.3% and 26.1%, respectively, and 30.2% compared with the combined injection group (see Figure 3 a, b). Immunofluorescence staining further confirmed the increase of CD11c-positive DCs in the GAZn-PEG group (see Figure 3 c). In addition, the secretion levels of proinflammatory cytokines IL-6, IL-12p70, and TNF-α in the GAZn-PEG-treated group were significantly increased, indicating the improvement of DCs maturation (see Figure 3 df).

[0075] In terms of T cell response, CD3 + T cells and CD69hiCD3 + The proportion of T cells increased significantly, indicating that T cells were effectively activated (see Figure 3 gi). The proportion of natural killer cells (NK cells) also showed a similar increasing trend, further confirming the activation effect of GAZn-PEG on the immune system (see Figure 3 l, m). In terms of tumor-associated macrophages (TAMs), CD80 + The proportion of TAMs increased, while CD206 + The decrease in the proportion of TAMs indicated that GAZn-PEG could change the phenotype of TAMs (see Figure 3 o, p). In contrast, regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) did not change significantly in the GAZn-PEG group, which further validated the specificity and effectiveness of our treatment strategy (see Figure 3 q, r).

[0076] Thus, GAZn-PEG nanoparticles exhibit excellent pharmacokinetic properties and can effectively activate the immune system, promote DCs maturation, T cell and NK cell infiltration, and regulate the phenotype of TAMs. These effects together promote effective anti-tumor immune responses, highlighting the potential of GAZn-PEG nanoparticles in cancer immunotherapy.

[0077] Encouraged by the significant effects of GAZn-PEG NPs in reducing HSP-90 expression and enhancing immune activation, we further explored the enhancement of pHIFU treatment by GAZn-PEG NPs in the U14 cervical cancer BALB / c mouse model. 3 -110mm3 The U14 tumor-bearing mice were randomly divided into five treatment groups: 1. PBS treatment only, 2. pHIFU treatment only, 3. GAZn-PEG NPs injection, 4. GA and Zn 2+ 5. GAZn-PEG NPs were injected and then pHIFU treatment was performed. On day 0 of the experiment, mice in groups 2-5 received a mixture of GA (2 mg / kg) and Zn 2+ Then, on the second day, mice in groups 2, 4, and 5 received pHIFU irradiation at 100 W for 6 seconds.

[0078] The results showed that the combined treatment of GAZn-PEG NPs and pHIFU completely inhibited tumor growth and achieved a 50% cure rate, while treatment with GAZn-PEG NPs or pHIFU alone showed only limited tumor inhibition effects. 2+ The median survival time of the combined treatment group was extended to 24 days, which was an improvement compared with pHIFU alone (18 days) and GAZn-PEG injection alone (18 days), but still lower than the 34 days of the GAZn-PEG NPs and pHIFU combined treatment group (see Figure 4 be).

[0079] To evaluate the early treatment effect, we collected tumor tissue sections on the second day after treatment and performed H&E staining and immunohistochemistry analysis, focusing on the degree of cell damage and the expression level of HSP-90. The results showed that the group treated with pHIFU after GAZn-PEG NPs injection (Group 5) showed the most extensive cell damage, while the other groups showed moderate damage. At the same time, the combined treatment group of GAZn-PEG NPs and pHIFU significantly reduced the expression of HSP-90, which was better than GA and Zn 2+ Mixed treatment group (see Figure 4 f). These findings confirm the potential of GAZn-PEG NPs in enhancing the therapeutic effect of pHIFU and inhibiting the expression of HSP-90, providing a new strategy for the immunotherapy of cervical cancer.

[0080] In order to further explore the anti-tumor immune response stimulated by the combined treatment of GAZn-PEG NPs and pHIFU, we performed the above five treatments on BALB / c mice bearing U14 tumors, and euthanized the mice on the 5th day after pHIFU treatment to collect tumor tissues for flow cytometry analysis. The results showed that the maturation of dendritic cells (DCs) in the combined treatment group of GAZn-PEG NPs and pHIFU was significantly improved, reaching an average of 43.05%, far exceeding the 30.8% of the pHIFU treatment group, the 33.4% of the GAZn-PEG treatment group alone, and the GA and Zn 2+ 37.6% of the combined treatment group (see Figure 5 a, b).

[0081] Compared with mice treated with only pHIFU, the total CD3 + T cells and CD69 expression increased significantly (see Figure 5 In addition, CD3 + CD4 + T cells and cytotoxic T cells (CD3 + CD8 + ) also increased significantly, which is consistent with the results of immunofluorescence staining (see Figure 5 fh). Combination therapy also significantly enhanced the secretion of cytotoxic IFN-γ, a key indicator of antitumor immune activation (see Figure 5 i, j). Notably, the combination therapy also significantly increased the number of cytotoxic NK cells and CD80 in tumors. + The number of M1-like macrophages and CD206 were decreased + The number of M2-like macrophages indicated that this treatment strategy had a significant effect on innate anti-tumor immunity (see Figure 5 kn).

[0082] Taken together these results, we confirmed that GAZn-PEG NPs combined with pHIFU therapy can together stimulate a strong antitumor immune response, as manifested by increased DC maturation, cytotoxic lymphocyte infiltration, and polarization of M2 to M1 macrophages. In addition, we evaluated the levels of various cytokines in tumors, which are characteristic of M1 and M2 macrophages, respectively. TNF-α (see Figure 5 o) and IFN-γ (see Figure 5 p) level was the highest, indicating that pHIFU treatment based on GAZn-PEG NPs can effectively activate the immune system.

[0083] Thus, GAZn-PEG NPs combined with pHIFU showed significant pharmacokinetic and immunological advantages in the cervical cancer model. This strategy not only significantly improved the therapeutic effect of pHIFU by reducing HSP-90 expression and enhancing the activation and infiltration of immune cells, but also established effective immune memory and prevented tumor recurrence. These findings further support the potential of GAZn-PEG NPs in cancer immunotherapy.

[0084] In summary, GAZn-PEG NPs can not only completely eliminate local tumors, but also continuously induce effective anti-tumor immune responses and inhibit cancer metastasis and recurrence, providing a simple and effective sensitization strategy for HIFU technology, promoting more effective HIFU technology treatment, and is expected to completely eradicate cancer.

[0085] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a zinc gamboge complex, characterized in that the steps include: S1. Mixing zinc ions and gambogic acid in a first organic solvent under stirring to obtain a first mixture; S2, adding triethylamine and polyvinyl pyrrolidone to the first mixture, stirring for a period of time to obtain a second mixture; S3, separating the precipitate in the second mixture by centrifugation, washing the obtained solid for multiple times and then vacuum drying to obtain the gambogic acid zinc complex.

2. The preparation method according to claim 1, characterized in that: In the step S1, under the stirring condition, the first solution containing the zinc ions is added dropwise to the second solution containing the gambogic acid to obtain the first mixture; Wherein, the solvents of the first solution and the second solution are both the first organic solvent.

3. The preparation method according to claim 1, characterized in that: In step S1, the stirring conditions include: a stirring speed of 1000-2000 rpm; or / and In the step S1, the molar ratio of the zinc ion to the gambogic acid is 1:(2-6); or / and In step S1, the first organic solvent is ethanol; or / and In step S2, the stirring time is 1-3 hours; or / and In step S3, the centrifugal conditions include: the centrifugal force is 3500g-4500g, and the centrifugal time is 2min-7min.

4. The preparation method according to any one of claims 1 to 3, characterized in that The steps also include: S4, modifying the surface of the solid obtained in step S3 with polyethylene glycol.

5. The preparation method according to claim 4, characterized in that: The step S4 comprises: S4-1, dispersing the solid obtained in step S3 in a second organic solvent, adding a third solution containing 1,2-dioleoyl-sn-glycero-3-phosphate, and performing ultrasonic treatment; S4-2, separating the gamboge acid zinc complex coated with 1,2-dioleoyl-sn-glycero-3-phosphate obtained in step S4-1 by centrifugation, washing it multiple times, dispersing it in the fourth solution, and stirring it at a certain stirring speed for a period of time to obtain a third mixture; Wherein, the solute of the fourth solution includes: cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-5000; S4-3, removing the solvent in the third mixture by rotary evaporation to obtain a zinc gamboge complex with surface modified with polyethylene glycol.

6. The preparation method according to claim 5, characterized in that: In the step S4-1, the second organic solvent is ethanol; or / and In the step S4-1, the solvent of the third solution is a third organic solvent; or / and In step S4-1, the conditions of the ultrasonic treatment include: the ultrasonic power is 50w-150w, and the ultrasonic time is 10min-20min; or / and In step S4-2, the centrifugal conditions include: a centrifugal force of 500g-1500g, a centrifugal time of 2min-7min; or / and In the step S4-2, the solvent of the fourth solution is a fourth organic solvent; or / and In step S4-2, the stirring speed is 1000-2000 rpm, and the stirring time is overnight; or / and In the step S4-3, the conditions for rotary evaporation include: providing a vacuum negative pressure environment, the temperature of rotary evaporation is 35°C-40°C, the speed of rotary evaporation is 50-100 rpm, and the time of rotary evaporation is 10min-20min.

7. The preparation method according to claim 6, characterized in that: In the step S4-1, the third organic solvent is chloroform; or / and In the step S4-2, the fourth organic solvent is chloroform.

8. A gambogic acid zinc complex prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the gambogic acid zinc complex as claimed in claim 8 in the preparation of a drug for treating cervical cancer.

10. Use of the gambogic acid zinc complex as claimed in claim 8 in preparing a system for treating cervical cancer, characterized in that: The system is capable of administering a therapeutically effective amount of the zinc gamboge acid complex to a patient in combination with applying high intensity focused ultrasound.

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